Evidence for a bidirectional element located downstream from the herpes simplex virus type 1 latency-associated promoter that increases its activity during latency

Abstract

Herpes simplex virus type 1 (HSV-1) latent infection in vivo is characterized by the constitutive expression of the latency-associated transcripts (LAT), which originate from the LAT promoter (LAP). In an attempt to determine the functional parts of LAP, we previously demonstrated that viruses harboring a DNA fragment 3' of the LAT promoter itself were able to maintain detectable promoter expression throughout latency whereas viruses not containing this element could not (J. R. Lokensgard, H. Berthomme, and L. T. Feldman, J. Virol. 71:6714-6719, 1997). This element was therefore called a long-term expression element (LTE). To further study the role of the LTE, we constructed plasmids containing a DNA fragment encompassing the LTE inserted into a synthetic intron between the reporter lacZ gene and either the LAT or the HSV-1 thymidine kinase promoter. Transient-expression experiments with both neuronal and nonneuronal cell lines showed that the LTE locus has an enhancer activity that does not activate the cytomegalovirus enhancer but does activate the promoters such as the LAT promoter and the thymidine kinase promoter. The enhancement of these two promoters occurs in both neuronal and nonneuronal cell lines. Recombinant viruses containing enhancer constructs were constructed, and these demonstrated that the enhancer functioned when present in the context of the viral DNA, both for in vitro infections of cells in culture and for in vivo infections of neurons in mouse dorsal root ganglia. In the infections of mouse dorsal root ganglia, there was a very high level of promoter activity in neurons infected with viruses bearing the LAT promoter-enhancer, but this decreased after the first 2 or 3 weeks. By 18 days postinfection, neurons harboring latent virus without the enhancer showed no beta-galactosidase (beta-gal) staining whereas those harboring latent virus containing the enhancer continued to show beta-gal staining for long periods, extending to at least 6 months postinfection, the longest time examined.

FIG. 1

Graphic map of the DNA structures of plasmids and viruses. (A) Plasmids were constructed by inserting either the CMV enhancer/promoter, the LAP, or the HSV-1 TK promoter upstream from the reporter gene lacZ. The presence of the LTE region and the synthetic intron are indicated by the LTE box and the splice donor (SD) and the splice acceptor (SA) sites, respectively. Arrows show the orientation of the inserted LTE or Sce (internal fragment of the I-SceI gene of S. cerevisiae) sequences. All transcriptional units were flanked with sequences from the HSV-1 gC gene, to allow the recombination at the gC locus of virus KOS dl1.8 HSV-1 (21), leading to KOS18, KOS22F, and KOS22R viruses. (B) Complete genome of HSV-1 and an expanded view of the long internal repeat (IRL). Positions on the HSV-1 genome as well as the flanking restriction sites of the LAP and LTE sequences used in this study are also indicated. The bottom line shows the region of the LAT intron (dark rectangle) and its overlap with the third exon of the ICP0 mRNA.

FIG. 1

Graphic map of the DNA structures of plasmids and viruses. (A) Plasmids were constructed by inserting either the CMV enhancer/promoter, the LAP, or the HSV-1 TK promoter upstream from the reporter gene lacZ. The presence of the LTE region and the synthetic intron are indicated by the LTE box and the splice donor (SD) and the splice acceptor (SA) sites, respectively. Arrows show the orientation of the inserted LTE or Sce (internal fragment of the I-SceI gene of S. cerevisiae) sequences. All transcriptional units were flanked with sequences from the HSV-1 gC gene, to allow the recombination at the gC locus of virus KOS dl1.8 HSV-1 (21), leading to KOS18, KOS22F, and KOS22R viruses. (B) Complete genome of HSV-1 and an expanded view of the long internal repeat (IRL). Positions on the HSV-1 genome as well as the flanking restriction sites of the LAP and LTE sequences used in this study are also indicated. The bottom line shows the region of the LAT intron (dark rectangle) and its overlap with the third exon of the ICP0 mRNA.

FIG. 2

β-Galactosidase activity from transfections of the CMV promoter plasmids. Dishes (60 mm) containing 10⁶ RS cells were transfected independently with 2 μg of DNA of plasmids pHB17.1, pHB30F, pHB12F, and pHB12R. At 2 days post-transfection, β-galactosidase activity was determined in vitro using CPRG as the substrate. Values are indicated as the mean of experiments conducted in triplicate and are expressed as fold increase over background.

FIG. 3

β-Galactosidase activity from transfections of the LAT or TK promoter plasmids. (A and B) Plasmids pHB18, pHB22F, pHB22R, and pHB23 were all or partly used to transfect RS (A) or ND7 (B) cells independently as described in Materials and Methods. (C) Similarly, plasmids pHB16, pHB19F, and pHB19R were transfected in RS cells. At 2 days posttransfection, β-galactosidase activity was measured using the CPRG assay. Values are indicated as the mean of experiments conducted in triplicate and are expressed as fold increase over background.

FIG. 4

β-Galactosidase activity from infected RS and ND7 cells in culture. RS and ND7 cells grown to near confluence were either mock infected or infected with KOS18, KOS22F, and KOS22R independently at a MOI of 3 PFU per cell. These viruses corresponded respectively to the insertion of plasmid pHB18, pHB22F, and pHB22R at the gC locus of the parental KOS dl1.8 HSV-1 (21). At 2 days p.i., cells were harvested and lysed and β-galactosidase activity was determined using the CPRG assay. Values are indicated as the mean of experiments conducted in triplicate and are expressed as fold increase over background.

FIG. 5

β-Galactosidase activity in whole-mount infected DRG by histochemical staining. Swiss Webster mice were inoculated with KOS18, KOS22F, and KOS22R, and DRG were removed and stained as indicated in Materials and Methods. L3, L4, and L5 DRG infected by KOS18 at 4 days p.i. (A) or 12 days p.i. (B), by KOS22F at 4 days p.i. (C) or 12 days p.i. (D), by KOS22R at 4 days p.i. (E) or 12 days p.i. (F), or by KOS22F at 6 months p.i. (G) are shown.

FIG. 5

β-Galactosidase activity in whole-mount infected DRG by histochemical staining. Swiss Webster mice were inoculated with KOS18, KOS22F, and KOS22R, and DRG were removed and stained as indicated in Materials and Methods. L3, L4, and L5 DRG infected by KOS18 at 4 days p.i. (A) or 12 days p.i. (B), by KOS22F at 4 days p.i. (C) or 12 days p.i. (D), by KOS22R at 4 days p.i. (E) or 12 days p.i. (F), or by KOS22F at 6 months p.i. (G) are shown.

FIG. 6

β-Galactosidase activity in infected DRG extracts by the quantitative CPRG assay. Swiss Webster mice were inoculated with KOS18, KOS22F, and KOS22R viruses. At the indicated time, cellular extracts from pooled L3, L4, L5, and L6 DRG were produced and used to quantify lacZ gene expression as indicated in Materials and Methods. Values are the mean of the DRG from three mice per time point per virus.

FIG. 7

Immunodetection of β-galactosidase (β-gal.) and HSV-1 antigens (Ag.) on infected DRG sections. Swiss Webster mice were inoculated with KOS22F and KOS22R viruses. At the indicated time, DRG were removed, equilibrated with 20% sucrose, embedded in OCT, and frozen in liquid nitrogen. Serial sections (6 μm), were cut, stained for β-galactosidase and HSV-1 antigens, and evaluated under a fluorescence microscope.